Various laboratory and clinical procedures employ biospecific affinity reactions. Such reactions are commonly employed in diagnostic testing of biological samples, or for the separation of a wide range of target substances, especially biological entities such as cells, proteins, nucleic acid sequences, and the like.
Various methods are available for analyzing or separating the above-mentioned target substances based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation may be employed for this purpose.
Small magnetic particles have proved to be quite useful in analyses involving biospecific affinity reactions, as they are conveniently coated with biofunctional polymers, e.g., proteins, provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents.
Small magnetic particles, such as those mentioned above, generally fall into two broad categories. The first category includes particles that are permanently magnetizable, or ferromagnetic; and the second comprises particles that demonstrate bulk magnetic behavior only when subjected to a magnetic field. The latter are referred to as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, materials exhibiting bulk ferromagnetic properties, e.g., magnetic iron oxide, may be characterized as superparamagnetic when provided in crystals of about 30 nm or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter due to strong particle-particle interaction.
U.S. Pat. No. 4,795,698 to Owen et al. relates to polymer-coated, sub-micron size colloidal superparamagnetic particles. The '698 patent describes the manufacture of such particles by precipitation of a magnetic species in the presence of a biofunctional polymer. The structure of the resulting particles, referred to herein as single-shot particles, has been found to be a micro-agglomerate in which one or more ferromagnetic crystallites having a diameter of 5-10 nm are embedded within a polymer body having a diameter on the order of 50 nm. These particles exhibit true colloidal behavior and do not exhibit an appreciable tendency to separate from aqueous suspensions for observation periods as long as several days.
Another method for producing superparamagnetic colloidal particles is described in U.S. application Ser. No. 07/397,106. In contrast to the particles described in the '698 patent, these latter particles are produced by directly coating a biofunctional polymer onto a pre-formed superparamagnetic crystallite. The resulting particles, referred to herein as DC particles, exhibit a significantly larger magnetic moment than single-shot particles of the same overall size.
Magnetic separation techniques are known wherein a magnetic field is applied to a fluid medium in order to separate ferromagnetic bodies from the fluid medium. In contrast, the tendency of colloidal superparamagnetic particles to remain in suspension, in conjunction with their relatively weak magnetic responsiveness, requires the use of high-gradient magnetic separation (HGMS) techniques in order to separate such particles from a fluid medium in which they are suspended. In HGMS systems, the gradient of the magnetic field, i.e. the spatial derivative, exerts a greater influence upon the behavior of the suspended particles than is exerted by the strength of the field at a given point.
HGMS systems can be divided into two broad categories. One such category includes magnetic separation systems which employ a magnetic circuit that is entirely situated externally to a separation chamber or vessel. Examples of such external separators are described in U.S. Pat. No. 5,186,827. In several of the embodiments described in the '827 patent, the requisite magnetic field gradient is produced by positioning permanent magnets around the periphery of a non-magnetic container such that the like poles of the magnets are in a field-opposing configuration. The extent of the magnetic field gradient within the test medium that may be obtained in such a system is limited by the strength of the magnets and the separation distance between the magnets. As mentioned in Cell Separation: Methods and Selected Applications, Pretlow and Pretlow eds., 1987, p. 262, in reference to external HGMS:
"If one then attempts to scale up the process by increasing the size of the vessel and the magnet similarly, one is often frustrated. The complex geometric aspects of magnetic field generation will not, in general, lead to gradients which still fill the same proportion of the new sample chamber."
Another type of HGMS separator utilizes a ferromagnetic collection structure that is disposed within the test medium in order to intensify an applied magnetic field and to produce a magnetic field gradient within the test medium. In one known type of internal HGMS system, fine steel wool or gauze is packed within a column that is situated adjacent to a magnet. The applied magnetic field is concentrated in the vicinity of the steel wires so that suspended magnetic particles will be attracted toward, and adhered to, the surfaces of the wires. One drawback of such systems is that the use of steel wool or gauze material can entrap non-magnetic components of the test medium by capillary action in the vicinity of intersecting wires or within interstices between intersecting wires. Thus, enlarging the separator serves to increase the amount of non-magnetic components that are so entrapped. Furthermore, an enlarged internal HGMS system, which consequently employs a greater bulk of ferromagnetic material, would suffer from mutual magnetic shielding of the ferromagnetic wire therein.
In both the internal and external HGMS apparatus described above, the limited extent of the high magnetic gradient within the test medium has hampered the ability to process large quantities of material in a conveniently rapid manner. Conventional HGMS separation techniques involve mixing colloidal magnetic particles with a test medium from which a target substance is to be separated. Then, the mixture is introduced into an HGMS apparatus as a single fluid. In order to obtain relatively rapid separations, it has been necessary to use small containers for the test medium in order to generate a sufficiently high gradient extending into the test medium. Increasing the size of the container increases the time required to perform a separation because the superparamagnetic particles most distant from the collection surface are then less responsive to the decreased gradient. Additionally, such particles must travel a greater distance through a relatively viscous fluid in order to be collected.
An internal separator is described in U.S. Pat. No. 5,200,084 employing loops of ferromagnetic wire that are spaced apart from each other. The loops are inserted into a test medium in order to generate an internal field gradient. As in the case of external HGMS systems, increasing the size of the apparatus while maintaining the separation distance between the ferromagnetic collecting wires would have the effect of decreasing the proportion of the volume within the separation chamber in which a high gradient field is present.
High gradient magnetic separation is useful for separating a wide variety of biological materials, including eucaryotic and procaryotic cells, viruses, nucleic acids, proteins and carbohydrates. In methods known heretofore, biological material has been separable by means of HGMS if it possesses at least one characteristic determinant, which is capable of being specifically recognized by and bound to a receptor, such as an antibody, antibody fragment, specific binding protein (e.g., protein A, streptavidin), lectin and the like.
High gradient magnetic separation is a method of choice for separating a cell subset of interest from a mixed population of eucaryotic cells, particularly if the subset of interest comprises but a small fraction of the entire population. Such separation generally relies upon the identification of cell surface antigens that are unique to a specific cell subset of interest. Antibodies directed to these antigens (sometimes referred to herein as "primary antibodies") are utilized directly, by attaching them to magnetic particles, or indirectly, by means of a second receptor/ligand interaction (e.g., avidin-coated magnetic particles recognizing biotinylated primary antibodies, or a second antibody that specifically recognizes an epitope of the primary antibody).
Cell separations are typically performed with one or both of the following objectives: (1) enrichment of a cell type of interest (often a rare cell type) from a mixed cell population; and/or (2) purging of an unwanted cell type (e.g., tumor cells, bacterial contaminants, and the like) from a mixed cell population. Two general approaches have been taken to accomplish one or both of these objectives. In one approach, the target cell types may be isolated by positive selection, wherein the cells of interest are labelled with antibodies and then separated from the remaining unlabelled cells in the population. In another approach, a cell type of interest may be isolated or enriched by "negative depletion," in which one or more unwanted cell types are labelled with antibodies and are then removed from the remaining unbound cell type. Depending on the desired outcome, these approaches have been used singly or in sequential combination.
The choice of positive, negative, or combination separation depends largely upon the subsequent use to which the separated cells will be put, as well as the limitations of currently-available magnetic separation methodologies described above. Positive selection is advantageous because of the relatively high yields and purity of the target cell suspensions obtained, often by means of single-step isolation methods. Positive selection is useful for separating target cells for purposes that do not require that the cells be fully functional, with respect to the availability and competence of cell-surface macromolecules. Such purposes include purging of unwanted cells, isolation of cells for further analysis (e.g., by flow cytometry), or identification of cells for diagnostic purposes. However, if the target cells are intended for re-introduction into a patient (e.g., hematopoietic stem cells from bone marrow or peripheral blood), or for other purposes in which full and unaltered biological functionality is required, it is desirable to obtain the target cells in their fully-functional, or native, condition. Since positively-selected cells are labelled with an antibody or other specific receptor, the target cells may be compromised by alterations or losses of the functionalities associated with the surface antigens to which the antibodies are bound. For example, many surface antigens are receptors that modulate activation of pleuripotent stem cells causing them to differentiate. If it is desired to obtain undifferentiated cells, subjecting the cells to treatment that causes them to differentiate, such as may occur during positive selection, can be counterproductive. As another example, many surface antigens, including the CD34 surface antigen associated with hematopoietic stem cells, are homing molecules that inform the cells where to migrate in the body after the cell has been reintroduced into a patient. Blocking such homing molecules with a binding agent during separation can hinder or prevent this critical specific localization process.
Another concern that arises from the attachment of antibody to cells which are to be re-introduced into a human patient relates to the fact that most monoclonal antibody used in immunological separation are raised in mice. A large proportion of the human population carries antibodies against mouse proteins, due to the common contamination of processed food products with mouse parts. As a result, introduction of mouse protein-bearing cells into a human patient can elicit an immune response which could impair the health and/or recovery of patients who may already be immunologically compromised.
Because of the undesirability of attaching labelling antibodies to certain target cell populations, several methods have been developed to remove these labelling antibodies from the cells. Such methods include (1) incubating isolated target cells with vast excesses of a competitive ligand to shift binding of the antibody from the cell surface to the ligand; (2) chemical methods, such as exposure to low pH or to various chaeotropic agents; and (3) treatment of isolated cells with proteolytic enzymes,
Each of the known treatments to remove labelling antibodies from positively selected cells can result in reduced target cell yield due to transfer steps or the rigors of the treatment. More importantly however, these methods have met with limited degrees of success in removal of antibodies from the target cells. Moreover, in the case of the enzymatic treatments, the propensity of the proteolytic enzyme to remove portions of the surface antigen to which the antibody is attached, or to non-specifically cleave other critical antigens on the cell surface, can cause additional undesirable results. For example, in Strauss et al., Am. J. Ped. Hematol. Oncol., 13: 217-221 (1991), chymopapain was found to destroy the epitope of the CD34 surface antigen that recognizes My-10 monoclonal antibodies.
Isolation of target cell types by negative depletion can be desirable because negative depletion avoids some of the undesirable results associated with labelling target cells with antibodies or other receptors. The ability to obtain native cells is important clinically in the isolation of hematopoietic cells, as described above, and may also be desirable in obtaining populations of other target cell types or substances. The major disadvantage of known negative depletion methods is that they typically result in low yield and low purity of the target cell type. This disadvantage is especially pronounced in separations of rare cell types, such as hematopoietic stem cells from bone marrow or peripheral blood, or fetal cells from maternal cell types in maternal blood.
Positive selection and negative depletion have been combined in attempts to increase the yield and purity of target cells. Although some measure of success may be obtained by a combined approach, the disadvantages associated with positive selection remain inherent in a two-step approach. Additional decreased yield and/or functionality can result from the added steps in the separation procedure.
It is apparent that HGMS affords certain advantages in performing separations based on biospecific affinity reactions involving colloidal magnetic particles. Nevertheless, currently available systems are limited with respect to the level of yields and purity achievable. Accordingly, a need exists for HGMS methods and devices that are simple, rapid, and reduce entrapment of non-target substances, thereby achieving both high yield and high purity. It would be of even greater advantage if such results could be obtained by a negative depletion method, wherein the cells or other substances of interest need not be attached to antibody or other receptors in order to be separated from a test medium. Such improvements in magnetic separation technology would clearly be of practical utility in conducting either laboratory- or clinical-scale separations, for diagnostic, therapeutic or preparative purposes.